Bone-promoting thermoresponsive macromolecules

ABSTRACT

Provided herein are injectable, thermoresponsive hydrogels that are liquid at room temperature, provide a carrier material, and gel at body temperature to allow for controlled release. In particular, PPCN-based hydrogels are provided with therapeutic agents (e.g., drugs, ions, etc.) incorporated within or appended thereto, and methods of preparation and use thereof, for example, for the promotion of bone formation/repair and/or the treatment of bone diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 16/468,224, filed on Jun. 10, 2019, now U.S. Pat. No.11,559,609, which is a § 371 U.S. National Entry Application based onPCT/US2017/065352, filed Dec. 8, 2017, which claims priority to U.S.Provisional Patent Application Ser. No. 62/432,266, filed Dec. 9, 2016,and to U.S. Provisional Patent Application Ser. No. 62/571,495, filedOct. 12, 2017, each of which are incorporated by reference in theirentireties.

SEQUENCE LISTING

The text of the computer readable sequence listing filed herewith,titled “35106-304_SEQUENCE_LISTING”, created Jan. 24, 2023, having afile size of 5,335 bytes, is hereby incorporated by reference in itsentirety.

FIELD

Provided herein are injectable, thermoresponsive hydrogels that areliquid at room temperature, provide a carrier material, and gel at bodytemperature to allow for controlled release. In particular, PPCN-basedhydrogels are provided with therapeutic agents (e.g., drugs, ions, etc.)incorporated within or appended thereto, and methods of preparation anduse thereof, for example, for the promotion of bone formation/repairand/or the treatment of bone diseases.

BACKGROUND

Significant focus in the field of bone tissue engineering has beenplaced on developing materials for repair of bone defects. Much lesswork has been directed toward bone diseases. As such, material strengthhas been the main focus rather than the material's ability to inducebone formation. These materials are often hard, filler pastes that arebiologically inert (e.g., methyl methacrylate) or brittle (e.g., calciumphosphate). Additionally, the challenges of inflammation andosteoporotic bone have been largely unexplored. An unmet need lies inthe development of materials that can be used with a minimally invasiveprocedure to conform to unique fracture sites, can interact with thecomplex cellular environment to accelerate healing in all bone types,induce stem cells to become bone cells, and allow for localized drugdelivery if necessary.

SUMMARY

Provided herein are injectable, thermoresponsive hydrogels that areliquid at room temperature, provide a carrier material, and gel at bodytemperature to allow for controlled release. In particular, PPCN-basedhydrogels are provided with therapeutic agents (e.g., drugs, ions, etc.)incorporated within or appended thereto, and methods of preparation anduse thereof, for example, for the promotion of bone formation/repairand/or the treatment of bone diseases.

In some embodiments, provided herein are compositions comprising aPPCN-based hydrogel comprising citric acid, poly(ethylene glycol),glycerol 1,3-diglycerolate diacrylate, and poly-(N-isopropylacrylamide)monomers and at least one bioactive agent incorporated therein and/orappended thereto. In some embodiments, the bioactive agent is anadditional monomer incorporated into the PPCN-based hydrogel backbone.In some embodiments, the bioactive agent is β-glycerophosphate. In someembodiments, the bioactive agent is a pendant group appended to a PPCNhydrogel. In some embodiments, the pendant group is a small molecule. Insome embodiments, the pendant group is a peptide. In some embodiments,the bioactive agent is a cyclic Arg-Gly-Asp (cRGD) peptide. In someembodiments, the bioactive agent is an ionic crosslinking agent. In someembodiments, the bioactive agent is Ca²⁺, Ba²⁺, or Sr^(2+.)

In some embodiments, provided herein are compositions comprising aPPCN-based phosphate-displaying hydrogel comprising citric acid,poly(ethylene glycol), glycerol 1,3-diglycerolate diacrylate,poly-(N-isopropylacrylamide), and β-glycerophosphate monomers. In someembodiments, the PPCN-based phosphate-displaying hydrogel comprises thestructure:

wherein x and y are independently 2-20. In some embodiments, thecomposition is prepared by (a) polycondensation of citric acid,poly(ethylene glycol), glycerol 1,3-diglycerolate diacrylate, andβ-glycerophosphate monomers; followed by (b) free radical polymerizationwith poly-(N-isopropylacrylamide). In some embodiments, the compositionis prepared by the reactions depicted in Scheme 1.

In some embodiments, provided herein are compositions comprising aPPCN-based peptide-displaying hydrogel comprising citric acid,poly(ethylene glycol), glycerol 1,3-diglycerolate diacrylate, andpoly-(N-isopropylacrylamide) monomers, and a peptide covalentlyconjugated to carboxy groups of the citric acid monomers. In someembodiments, the peptide is covalently conjugated via carbodiimidechemistry to carboxy groups of the citric acid monomers. In someembodiments, the peptide is cyclic Arg-Gly-Asp (cRGD). In someembodiments, the cRGD is covalently conjugated to carboxy groups ofPPCN. In some embodiments, the composition is prepared by reactionsdepicted in Scheme 2.

In some embodiments, provided herein are compositions comprising aPPCN-based hydrogel comprising citric acid, poly(ethylene glycol),glycerol 1,3-diglycerolate diacrylate, and poly-(N-isopropylacrylamide)monomers, and metal ion crosslinks. In some embodiments, compositionscomprise metal-ion-crosslinked PPCN. In some embodiments, compositionsare prepared by incubating the PPCN in the presence of a salt of themetal ion. In some embodiments, the metal ion is Ca²⁺, Ba²⁺, or Sr^(2+.)

In some embodiments, provided herein are methods of facilitating bonerepair comprising administering a composition described herein (e.g., aPPCN-based hydrogel) to fractured or diseased bone site, and allowingthe composition to gel.

In some embodiments, provided herein is the use of a compositiondescribed herein (e.g., a PPCN-based hydrogel) to facilitate bonerepair.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 . (a) MALDI spectrum of PPCN-cRGD major peaks include expectedionized cyclic-RGDfC at 581.7 m/z and full monomer at 1156 m/z. (b)ICP-OES of 50 mM and 100 mM Sr²⁺ release from PPCN-Sr gels revealsmajority of strontium is released in 1 week. FT-IR of PPCN-phos showsgrowth of peaks associated with reagent β-glycerophosphate, namely thebroadening of the OH stretch at 3400 nm⁻¹ and growth of a new peakaround 1238 nm⁻¹ attributed to phosphonate. Elemental analysiscorroborates this data with a small reduction in carbon due to thedisplacement of carbon-rich PEG chains by β-glycerophosphate.

FIG. 2 . Rheological characterization of PPCN (a) as compared toPPCN-cRGD (b), PPCN-Sr (c) and PPCN-phos (d) confirms that all threeosteoinductive variants maintain thermoresponsive behavior and exhibit alower LCST transition than PPCN.

FIG. 3 . LIVE/DEAD imaging of hMSCs and MC3T3s seeded in 3D gels andcultured in regular DMEM. Cells were seeded in either alginate, PPCN,PPCN-cRGD, PPCN-Sr, or PPCN-phos gels. Cells grown in PPCN-cRGD gelsshow spreading morphology confirming the functionality of RGD ispreserved post-conjugation. Viability in all gels is above 75%. Imageswere taken at day 10 and representative images shown from n=3 wells.

FIG. 4 . Day 3 and 10 alkaline phosphatase (ALP) activity shown for (a)hMSCs and (b) MC3T3s. No significant increase in ALP is detected forhMSCs at day 10. However, significant increase in ALP is detected forMC3T3s grown in PPCN-Sr and PPCN-phos gels. *P-value<0.05 and**P-value<0.01.

FIG. 5 . Day 21 characterization of hMSCs via staining for calciumdeposition by Alizarin Red S, immunohistochemistry for osteopontin(OPN), and osteocalcin (OCN). Corresponding quantification is shown onthe right. Mineralization is seen in all three functionalizations. Fromthe data, osteopontin expression is highest in PPCN-Sr and osteocalcinexpression is highest in PPCN-phos.

FIG. 6 . MicroCT analysis of intramuscular femoral injection of PPCN andPPCN-Sr is shown from day 0 to day 42. The top right panel orients themicroCT images onto the mouse femur. The bottom right panel shows thequantification of the microCT images, n=3. Mineralization is observed inthe PPCN-Sr group beginning at day 10 and increasing consistentlythrough the 6 week period. No mineralization is observed in thenon-functionalized PPCN control. Mineralized regions were quantifiedwith Osirix by threshold intensity and reconstructed to show the 3Dinlay.

FIG. 7 . PPCN-Sr demonstrates significant osteocalcin expression andcell infiltration (b) as compared to PPCN control (a). Alizarin Red Sstaining for mineralization also demonstrates robust mineralization inPPCN-Sr (d) as detected by red calcium deposits and compared tonon-functionalized PPCN (c). Full tissue section is shown for PPCN andPPCN-Sr in (e) and (f), respectively.

FIG. 8 . Day 42 ICP analysis of 8 mouse organs shows that strontium isnot present in any of the main organs upon digestion (a). Additionally,XPS analysis of sectioned muscle tissue show that strontium is clearedand no longer present at the site of injection (b). XPS analysis showscalcium, phosphate and oxygen content consistent with mineralization inthe PPCN-Sr group, but no local strontium remaining at week 6.

DEFINITIONS

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of embodimentsdescribed herein, some preferred methods, compositions, devices, andmaterials are described herein. However, before the present materialsand methods are described, it is to be understood that this invention isnot limited to the particular molecules, compositions, methodologies orprotocols herein described, as these may vary in accordance with routineexperimentation and optimization. It is also to be understood that theterminology used in the description is for the purpose of describing theparticular versions or embodiments only, and is not intended to limitthe scope of the embodiments described herein.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. However, in case of conflict,the present specification, including definitions, will control.Accordingly, in the context of the embodiments described herein, thefollowing definitions apply.

As used herein and in the appended claims, the singular forms “a”, “an”and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a polymer” is a reference toone or more polymers and equivalents thereof known to those skilled inthe art, and so forth.

As used herein, the term “and/or” includes any and all combinations oflisted items, including any of the listed items individually. Forexample, “A, B, and/or C” encompasses A, B, C, AB, AC, BC, and ABC, eachof which is to be considered separately described by the statement “A,B, and/or C.” As used herein, the term “comprise” and linguisticvariations thereof denote the presence of recited feature(s),element(s), method step(s), etc. without the exclusion of the presenceof additional feature(s), element(s), method step(s), etc. Conversely,the term “consisting of” and linguistic variations thereof, denotes thepresence of recited feature(s), element(s), method step(s), etc. andexcludes any unrecited feature(s), element(s), method step(s), etc.,except for ordinarily-associated impurities. The phrase “consistingessentially of” denotes the recited feature(s), element(s), methodstep(s), etc. and any additional feature(s), element(s), method step(s),etc. that do not materially affect the basic nature of the composition,system, or method. Many embodiments herein are described using open“comprising” language. Such embodiments encompass multiple closed“consisting of” and/or “consisting essentially of” embodiments, whichmay alternatively be claimed or described using such language.

As used herein, the term “substantially all,” “substantially complete”and similar terms refer to greater than 99%; and the terms“substantially none,” “substantially free of,” and similar terms referto less than 1%.

The term “about” allows for a degree of variability in a value or range.As used herein, the term “about: refers to values within 10% of therecited value or range (e.g., about 50 is the equivalent of 45-55).

As used herein, the term “polymer” refers to a chain of repeatingstructural units or “monomers”, typically of large molecular mass.Examples of polymers include homopolymers (single type of monomersubunits), copolymers (two types of monomer subunits), andheteropolymers (e.g., three or more types of monomer subunits). As usedherein, the term “oligomer” refers to a polymer of only a few monomerunits (e.g., 2, 3, 4, 5, or more) up to about 50 monomer units, forexample a dimer, trimer, tetramer, pentamer, hexamer . . . decamer, etc.

As used herein, the term “linear polymer” refers to a polymer in whichthe molecules form long chains without branches or crosslinkedstructures.

As used herein, the term “branched polymer” refers to a polymercomprising a polymer backbone with one or more additional monomers, orchains or monomers, extending from polymer backbone. The degree ofinterconnectedness of the “branches” is insufficient to render thepolymer insoluble.

As used herein, the term “pre-polymer” refers to linear or branchedpolymers (e.g., not significantly crosslinked) that have the capacity tobe crosslinked under appropriate conditions (e.g., to “cure” and/or forma thermoset or hydrogel), but have not been subjected to the appropriateconditions.

As used herein, the term “crosslinked polymer” refers to a polymer witha significant degree of interconnectedness between multiple polymerstrands, the result of which is an insoluble polymer network. Forexample, multiple polymer stands may be crosslinked to each other atpoints within their structures, not limited to the ends of the polymerchains.

As used herein, the term “biocompatible” refers to materials, compounds,or compositions means that do not cause or elicit significant adverseeffects when administered to a subject. Examples of possible adverseeffects that limit biocompatibility include, but are not limited to,excessive inflammation, excessive or adverse immune response, andtoxicity.

As used herein, the term “hydrogel” refers to a three-dimensional (3D)crosslinked network of hydrophilic polymers that swells, rather thanbeing dissolved, in water.

As used herein, the term “thermoresponsive” refers to material thatexhibit altered physical characteristics at different temperatureranges. Particularly relevant herein are “phase-transitioningthermoresponsive” materials. Phase-transitioning thermoresponsive”materials are soluble or in a liquid state at a first temperature range(e.g., below 26° C.) and insoluble or in a solid state at a secondtemperature range (e.g., 30-45° C.).

DETAILED DESCRIPTION

Provided herein are injectable, thermoresponsive hydrogels that areliquid at room temperature, provide a carrier material for ions ortherapeutics, and gel at body temperature to allow for controlledrelease. In some embodiments, materials are antioxidant and can promotebone formation based on material properties, without osteogenicsupplementation. In some embodiments, materials comprise, for example,bone-promoting agents, such as phosphate, cRGD, and/or strontium.

In some embodiments, provided herein are thermoresponsive hydrogelscomprising citric acid, poly(ethylene glycol), glycerol1,3-diglycerolate diacrylate, and poly-(N-isopropylacrylamide) monomers.In some embodiments, thermoresponsive hydrogels are PPCN-basedhydrogels. In some embodiments, a PPCN-based hydrogel comprises citricacid, poly(ethylene glycol), glycerol 1,3-diglycerolate diacrylate, andpoly-(N-isopropylacrylamide) monomers and one or more additional monomergroups (e.g., β-glycerophosphate). In some embodiments, a PPCN-basedhydrogel comprises citric acid, poly(ethylene glycol), glycerol1,3-diglycerolate diacrylate, and poly-(N-isopropylacrylamide) monomersand is conjugated to one or more bioactive agents (e.g., metal ions(e.g., Sr²⁺), peptides (e.g., cRGD), etc.).

In some embodiments, negatively-charged carboxyl groups on thePPCN-based hydrogels (e.g., displayed on the citric acid monomers)conjugate positively charged metal ions (e.g., Sr²⁺, Ca²⁺, Ba²⁺, etc.).In some embodiments, coordination of metal ions by separate carboxylgroups results in crosslinks in the PPCN-based polymer. In someembodiments, materials herein are not limited by the identity of themetal ion. In some embodiments, metal ions are introduced as salts tothe PPCN-based materials.

In some embodiments, carboxyl groups on the PPCN-based hydrogels (e.g.,displayed on the citric acid monomers) are conjugated via appropriatelinker chemistry to peptides or bioactive small molecules. In someembodiments, suitable chemistires for linking bioactive peptides and/orsmall molecules to the PPCN-based hydrogels include alkyne/azide,thiol/maleimide, thiol/haloacetyl (e.g., iodoacetyl, etc.),thiol/pyridyl disulfide (e.g. pyridyldithiol, etc.), sulphonylazides/thio acids, etc.

In some embodiments, a bioactive peptide that facilitate bone healingand/or repair is conjugated to a PPCN-based hydrogel. Suitable peptidesinclude the P-15 peptide (Bhatnagar et al. Tissue Eng.1999; 5(1):53-65.;incorporated by reference in its entirety), an RGD containing peptide(Ruoslahti & Pierschbacher. Cell. 1986; 44(4):517-8.; incorporated byreference in its entirety), GFOGER(glycine-phenylalanine-hydroxyproline-glycine-glutamate-arginine) (SEQID NO: 1), collagen-binding motif (CBM), DGEA (Asp-Gly-Glu-Ala) (SEQ IDNO: 2), SVVYGLR (Ser-Val-Val-Tyr-Gly-Leu-Arg) (SEQ ID NO: 3), KRSR(lysine-arginine-serine-arginine) (SEQ ID NO: 4), FHRRIKA(Phe-His-Arg-Arg-Ile-Lys-Ala) (SEQ ID NO: 5), Fibronectin (FN)-derivedpeptides, and other ECM-derived peptides (Pountos et al. BMC Med. 2016;14: 103.; incorporated by reference in its entirety).

In some embodiments, PPCN or a PPCN-based polymer or hydrogel comprisescomprise at least 0.1% citric acid monomers(e.g., >0.1%, >0.2%, >0.5%, >1%, >2%, >3%, >4%, >5%, >10%, >20%, >30%, >40%, >50%, >60%, >70%, >80%, >90%, >95%, >98%, >99%).In some embodiments, polymers herein comprise less than 99% citric acidmonomers (e.g., <99%, <98%, <95%, <90%, <80%, <70%, <60%, <50%, <40%,<30%, <20%,<10%, <5%,<4%, <3%, <2%, <1%, <0.5%,). In some embodiments,polymers comprise about 99%, about 98%, about 95%, about 90%, about 80%,about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about10%, about 5%, about 4%, about 3%, about 2%, about 1%, or about 0.5%citric acid monomers.

In some embodiments, PPCN or a PPCN-based polymer or hydrogel comprisescomprise at least 0.1% polyethylene glycol monomers(e.g., >0.1%, >0.2%, >0.5%, >1%, >2%, >3%, >4%, >5%, >10%, >20%, >30%, >40%, >50%, >60%, >70%, >80%, >90%, >95%, >98%, >99%).In some embodiments, polymers herein comprise less than 99% polyethyleneglycol monomers (e.g., <99%, <98%, <95%, <90%, <80%, <70%, <60%, <50%,<40%, <30%, <20%,<10%, <5%,<4%, <3%, <2%, <1%, <0.5%,). In someembodiments, polymers comprise about 99%, about 98%, about 95%, about90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%,about 20%, about 10%, about 5%, about 4%, about 3%, about 2%, about 1%,or about 0.5% polyethylene glycol monomers.

In some embodiments, PPCN or a PPCN-based polymer or hydrogel comprisesat least 0.1% glycerol 1,3- diglycerolate diacrylate monomers(e.g., >0.1%, >0.2%, >0.5%, >1%, >2%, >3%, >4%, >5%, >10%, >20%, >30%, >40%, >50%, >60%, >70%, >80%, >90%, >95%, >98%, >99%).In some embodiments, polymers herein comprise less than 99% glycerol1,3- diglycerolate diacrylate monomers (e.g., <99%, <98%, <95%, <90%,<80%, <70%, <60%, <50%, <40%, <30%, <20%,<10%, <5%,<4%, <3%, <2%, <1%,<0.5%). In some embodiments, polymers comprise about 99%, about 98%,about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about40%, about 30%, about 20%, about 10%, about 5%, about 4%, about 3%,about 2%, about 1%, or about 0.5% glycerol 1,3-diglycerolate diacrylatemonomers.

In some embodiments, PPCN or a PPCN-based polymer or hydrogel comprisesat least 0.1% N-isopropylacrylamide monomers(e.g., >0.1%, >0.2%, >0.5%, >1%, >2%, >3%, >4%, >5%, >10%, >20%, >30%, >40%, >50%, >60%, >70%, >80%, >90%, >95%, >98%, >99%).In some embodiments, polymers herein comprise less than 99%N-isopropylacrylamide monomers (e.g., <99%, <98%, <95%, <90%, <80%,<70%, <60%, <50%, <40%, <30%, <20%,<10%, <5%,<4%, <3%, <2%, <1%, <0.5%).In some embodiments, polymers comprise about 99%, about 98%, about 95%,about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about30%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2%, about1%, or about 0.5% N-isopropylacrylamide monomers.

In some embodiments, materials described herein comprise composites ofthe PPCN-based thermoresponsive hydrogel materials described herein andone or more additional components. In some embodiments, additionalcomponents comprise 1-99 wt % of the composite material (e.g., 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, orranges therebetween). In some embodiments, a composite materialcomprises at least 1%(e.g., >>1%, >2%, >3%, >4%, >5%, >10%, >20%, >30%, >40%, >50%, >60%, >70%, >80%, >90%, >95%, >98%, >99%)PPCN-based hydrogel. In some embodiments, a thermoresponsive compositematerial comprises less than 99% (e.g., <99%, <98%, <95%, <90%, <80%,<70%, <60%, <50%, <40%, <30%, <20%, <10%, <5%,<4%, <3%, <2%, <1%)PPCN-based hydrogel. In some embodiments, a composite material comprisesa PPCN-based hydrogel in an amount of about 99%, about 98%, about 95%,about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about30%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2%, about1%, or ranges therebetween. The aforementioned percentages may be wt %or molar %.

In some embodiments, composites of thermoresponsive PPCN-based hydrogelmaterials and a bioceramic component are provided. Suitable bioceramicsinclude hydroxyapatite (HA; Ca₁₀(PO₄)₆(OH)₂), tricalcium phosphate beta(β TCP; Ca₃(PO₄)₂), and mixtures of HAP and β TCP.

In some embodiments, composite materials comprise a PPCN-based hydrogeland one or more additional polymeric components. Suitable biodegradeablepolymers include, but are not limited to: collagen, elastin, hyaluronicacid and derivatives, sodium alginate and derivatives, chitosan andderivatives gelatin, starch, cellulose polymers (for examplemethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose,carboxymethylcellulose, cellulose acetate phthalate, cellulose acetatesuccinate, hydroxypropylmethylcellulose phthalate), casein, dextran andderivatives, polysaccharides, poly(caprolactone), fibrinogen,poly(hydroxyl acids), poly(L-lactide) poly(D,L lactide),poly(D,L-lactide-co-glycolide), poly(L-lactide-co-glycolide), copolymersof lactic acid and glycolic acid, copolymers of ε-caprolactone andlactide, copolymers of glycolide and ε-caprolactone, copolymers oflactide and 1,4-dioxane-2-one, polymers and copolymers that include oneor more of the residue units of the monomers D-lactide, L-lactide,D,L-lactide, glycolide, ε-caprolactone, trimethylene carbonate,1,4-dioxane-2-one or 1,5-dioxepan-2-one, poly(glycolide),poly(hydroxybutyrate), poly(alkylcarbonate) and poly(orthoesters),polyesters, poly(hydroxyvaleric acid), polydioxanone, poly(ethyleneterephthalate), poly(malic acid), poly(tartronic acid), polyanhydrides,polyphosphazenes, poly(amino acids), and copolymers of the abovepolymers as well as blends and combinations of the above polymers. (Seegenerally, Illum, L., Davids, S. S. (eds.) “Polymers in Controlled DrugDelivery” Wright, Bristol, 1987; Arshady, J. Controlled Release 17:1-22,1991; Pitt, Int. J. Phar. 59:173-196, 1990; Holland et al., J.Controlled Release 4:155-0180, 1986; herein incorporated by reference intheir entireties). Suitable non-biogregradable polymers include siliconerubber, polyethylene, acrylic resins, polyurethane, polypropylene, andpolymethylmethacrylate.

As described throughout, provided herein are PPCN-based materialscomprising curable (e.g., thermoresponsive) hydrogels and/or andcomposites thereof. These materials find use in a variety ofapplications. For example, any application in which it is desired that amaterial be applied in a liquid and/or soluble form, and then is(rapidly) rendered solid and/or insoluble when exposed to desiredconditions (e.g., physiological temperature). Materials described hereinfind use, for example, in medical and dental bone repair applications,such as, repair of craniofacial injuries, stabilizing complex fractures,promoting bone growth, bone regeneration, as a bone-void filler,adhering implants, etc. In some embodiments, materials find use innon-medical/dental applications. In some embodiments, the PPCN-basedmaterials described herein are liquid at sub-physiologic temperatures(e.g., 36° C., 35° C., 34° C., 33° C., 32° C., 31° C., 30° C., 29° C.,28° C., 27° C., 26° C., 25° C., 24° C., 23° C., 22° C., 21° C., 20° C.,or lower or ranges therebetween). In some embodiments, the PPCN-basedmaterials described herein gel at or near physiologic temperatures(e.g., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C.,38° C., 39° C., 40° C., or ranges therebetween).

In some embodiments in which the materials herein are used for therepair, stabilization, regeneration, growth, etc. of bone or bonefractures/injuries, the materials further comprise additionalcomponents/agents to facilitate incorporation into bone, bone growth,bone regeneration, etc. In some embodiments, additionalcomponents/agents are incorporated into the materials and aresubsequently encapsulated within the material upon curing. In suchembodiments, additional components/agents are non-covalently associatedwith the PPCN-based hydrogels and other components of the materials. Inother embodiments, additional components/agents are covalently-linkedPPCN-based hydrogel.

In some embodiments, the materials described herein find use in thedelivery of growth factors or other bioactive agents for the repair ofbone defects and/or regeneration of bone. Suitable agents for use inembodiments herein include bone morphogenic proteins (e.g., BMP-1,BMP-2, BMP-4, BMP-6, and BMP-7); members of the transforming growthfactor beta (TGF-β) superfamily including, but not limited to, TGF-β1,TGF-β2, and TGF-β3; growth differentiation factors (GDF1, GDF2, GDF3,GDFS, GDF6, GDF7, myostatin/GDF8, GDF9, GDF10, GDF11, and GDF15);vascular endothelial growth factor (VEGF); fibroblast growth factor(FGF); etc. These agent, or others, may be covalently linked tomaterials described herein or components thereof, non-covalentlyassociated with moieties displayed on materials described herein orcomponents thereof, embedded within materials described herein, etc.

In some embodiments, the PPCN-based hydrogels herein find use as acarrier for therapeutics (e.g., osteoporotic drugs), cells (e.g., stemcells), growth factors, etc. In some embodiments, the PPCN-basedhydrogels herein provide controlled, localized delivery of bioactiveagents encapsulated therein.

In some embodiments, provided herein are methods comprisingadministering a composition comprising a PPCN-based hydrogel describedherein to a bone defect or fracture. In some embodiments, providedherein is the use of a PPCN-based hydrogel described herein to repair abone defect or fracture.

In some embodiments, the PPCN-based hydrogels described herein areinjected into a fracture site (e.g., during orthopedic surgery) tofacilitate and/or accelerate healing.

Experimental Cell Culture

For gel studies, human mesenchymal stem cells (hMSCs, ATCC) werecultured in Dulbecco's Modified Eagle medium (DMEM) and supplementedwith 10% FBS and 5 ml 10× penicillin-streptomycin with no furtherosteogenic supplementation. hMSCs used in these studies were at passage6 or below. Murine pre-osteoblast MC3T3-E1 cells (ATCC) were cultured inDMEM:F12. Both cell lines were cultured at 37° C. and 5% carbon dioxide(CO₂).

Materials Preparation & Characterization

PPCN (poly (polyethylene glycol citrate-co-N-isopropylacrylamide) wasprepared by polycondensation and subsequent radical polymerization.

To prepare PPCN-phos, the original PPCN synthesis was adjusted to add0.1 molar ratio of β-glycerophosphate in the first polycondensation step(Scheme 1). The subsequent reaction steps were unchanged.

-   -   Scheme 1. Synthetic scheme of PPCN-phos synthesis.        β-glycerophosphate was added during step 1 (polycondensation) of        PPCN synthesis in 0.1 or 0.2 molar ratios. The proposed        locations of phosphate attachment are shown—specifically via        reaction between the reactive hydroxyls of β-glycerophosphate        and the available carboxyl groups of citric acid. PPCN-phos of        0.1 molar ratio was used in subsequent cell studies because        higher molar ratios exhibited overcrosslinking.

To prepare PPCN-cRGD gels, PPCN was formed as reported. Then, cyclicRGDfC peptide (ABI Scientific) was covalently conjugated via maleimidechemistry to the available carboxy groups of citric acid within the PPCNpolymer chain (Scheme 2). The intended density of cyclic RGD peptide was10%.

-   -   Scheme 2. Synthetic scheme of PPCN-Sr and PPCN-cRGD syntheses.        PPCN was prepared by polycondensation and subsequent free        radical polymerization as previously reported. Then, PPCN-cRGD        gels were prepared by covalently conjugating cyclic RGDfC        peptide via maleimide chemistry to the available carboxylic acid        groups of citric acid within the PPCN polymer chain. To prepare        PPCN-Sr gels, PPCN was dissolved in PBS (1×) at 100 mg/ml. 100        mM of SrCl₂ 6H₂O (Sigma) was mixed into PPCN/PBS solutions and        left to crosslink overnight at 4° C. prior to use.

To prepare PPCN-Sr gels, PPCN was dissolved in PBS (1×) at 100 mg/ml.100 mM of SrCl₂ 6H₂O (Sigma) was mixed into PPCN/PBS solutions and leftto crosslink overnight at 4° C. (Scheme 2).

Characterization for gels is shown in FIG. 1 . PPCN-cRGD conjugation wasconfirmed by matrix assisted laser desorption ionization (MALDI) (FIG. 1a ). The samples were prepared with an alpha-cyano-4-hydroxycinnamicacid matrix. PPCN-Sr was characterized by a strontium release study viainductively coupled plasma optical emission spectroscopy (Thermo iCAP7600 ICP-OES). Gels were immersed in simulated body fluid and solutionwas removed over a 14 day time course and assayed for Sr²⁺ concentration(FIG. 1 b ). PPCN-phos was characterized by FT-IR (Bruker Tensor 37) andXPS (FIG. 1 c ). Further rheological characterization was carried out ona TA instruments DHR rheometer with a 20 mm 2° cone peltier plategeometry and solvent trap cover to minimize sample evaporation. Gelswere studied in a temperature ramp experiment from 15° C. to 45° C. witha heating rate of 5° C./min. The viscoelastic moduli were monitored atan applied angular frequency of 10 rad/s and strain amplitude of 5%. Agap height of 52 μm was used for all samples. The initial change inviscoelastic properties was characterized by an increase of storagemodulus (G′) over loss modulus (G″).

3D Differentiation Studies

Cells were encapsulated in various thermoresponsive PPCN solutions of100 mg/mL PPCN in PBS (1×). Cells were added to solution via uniformmixing at a concentration of 1×10⁵ cells/mL liquid PPCN. The cell-PPCNmixture was incubated at 37° C. for 5 min to allow gels to form. Oncethe gel was formed, warm media was added on top and changed every 2days. Prior to seeding, the plate was coated with Sigma-cote to preventcell attachment to the plate, ensuring that all cells that persistthrough media changes persist within the 3D gel environment. Duringhandling, the plates were kept on a plate-warmer to ensure the gel wouldnot be dissolved or diluted as a result of temperature fluctuations. Ateach time-point, the media was removed and the gel was collected andfrozen in the case of ALP and DNA analysis or assayed directly in thecase of LIVE/DEAD, alizarin red staining, and immunohistochemistry.

Osteodifferentiation Assays

For early detection of osteodifferentiation (FIG. 4 ), alkalinephosphatase (ALP) was measured. The gels were collected at day 3 and day10 and extracellular alkaline phosphatase (ALP) activity was detected bya fluorometric kit (Biovision). A non-fluorescent substrate,4-Methylumelliferyl phosphate disodium salt (MUP), was added and cleavedby ALP, which results in a fluorescent signal (Ex/Em=360/440 nm). Thefluorescence was read on a micro-plate reader. The enzymatic activitywas calculated based on serially diluted gel standards and normalized tototal DNA content with a concurrent Quant-iT PicoGreen assay (ThermoFisher).

For late detection of osteodifferentiation (FIG. 5 ), Alizarin Red Sstaining and immunohistochemistry for osteocalcin (OCN) and osteopontin(OPN) was performed. For Alizarin Red S, the cells were fixed inside thewarmed gels and stain was allowed to permeate gels while excess stainwas subsequently rinsed out with several washes of DI water.Mineralization was visualized with light microscopy. Forimmunohistochemistry, the cells were fixed inside the warmed gels andprimary antibodies for OPN or OCN were added and counterstained withDAPI.

Mineralization

To assess mineralization, mice were anesthetized with isoflurane andplaced on the heated microCT bed. Images were acquired with apreclinical microPET/CT imaging system, nanoScan scanner (Mediso-USA,Boston, Mass.). Data were acquired with “medium” magnification, 33 μmfocal spot, 1×1 binning, with 720 projection views over a full circle,with a 300 ms exposure time. Images were acquired using 35 kVp. Theprojection data was reconstructed with a voxel size of 68 μm usingfiltered back-projection software from Mediso. The reconstructed datawere visualized and segmented in Osirix Lite for Mac. Using the coronalplane, images were quantified by creating regions of interest (ROI) with2D region-growing using a lower threshold of 600 and an upper thresholdof 10,000 Hounsfield units (HU). The regions of interests (ROIs) wereused for quantification of mineralization by calculating the mean HU foreach ROI (bone is 700 to 3,000 HU).

Immunofluorescence

Animals were euthanized by carbon dioxide inhalation. Tissues werecollected and fixed by 4% paraformaldehyde in PBS overnight at 4° C.Samples were washed by PBS with several changes to removeparaformaldehyde residue. The samples were dehydrated by series ethanol,cleared by xylene, and embedded in paraffin. Sections of 5 micronthickness were cut and mounted on slides. Sections were treated byxylene to remove paraffin, hydrated by alternating ethanol and water.Slides were immersed into antigen retrieval buffer (10 mM Sodiumcitrate, 0.05% Tween 20, pH 6.0) and heated at 100° C. for 15 min. Afterwashed by PBS, the samples were blocked by 5 mg/mL BSA, 5% normal goatserum in PBS for 30 min. Samples were incubated with primary antibodydiluted in blocking buffer at 4° C. overnight. Slides were washed by PBS3×5 min, then incubated with secondary antibody diluted by blockingbuffer at room temperature for 30 min. Slides were washed by PBS 6×5min, then mounted with anti-fade medium and sealed by nail polish.Images were taken by Nikon TE-2000U microscopy or Cytation5 imagereader.

Strontium Distribution

Strontium distribution was assayed in several main organs namely theheart, brain, spleen, testes, muscle, lung, kidney, and liver. Aftereuthanization, each organ was collected and stored in −80° C. untilanalysis. Tissue digestion was carried out by adding 70% nitric acid and37% hydrogen peroxide to each sample. Samples were uncapped after 1 hourto release built up gas. Tissues were left to digest for 2 days at roomtemperature. After 2 days, the samples were diluted down to 2.4% acid inMQ water and prepared for ICP-OES as mentioned above.

Elemental Analysis

XPS analysis of the sectioned tissue was conducted on a Thermo FisherESCALab 250Xi using Al K-alpha X-ray source (1486.6 eV) (Thermo FisherScientific, Waltham Mass.). The monochromated X-ray beam spot size was300 μm in diameter and the power was 100 watts. A pass energy of 100 eVand step size of 1 eV were used for the survey scan. For the highresolution scan, 50 eV of pass energy and a 0.1 eV step size were used.The dwell time was 50 ms. The XPS spectra were calibrated withadventitious carbon peak at 284.8 eV. All XPS data were processed withAvantage software.

1. A composition comprising a PPCN-based hydrogel comprising citricacid, poly(ethylene glycol), glycerol 1,3-diglycerolate diacrylate, andpoly-(N-isopropylacrylamide) monomers and at least one bioactive agentincorporated therein and/or appended thereto.
 2. The composition ofclaim 1, wherein the bioactive agent is an additional monomerincorporated into the PPCN-based hydrogel backbone.
 3. The compositionof claim 2, wherein the bioactive agent is β-glycerophosphate.
 4. Thecomposition of claim 1, wherein the bioactive agent is a pendant groupappended to a PPCN hydrogel.
 5. The composition of claim 4, wherein thependant group is a small molecule.
 6. The composition of claim 4,wherein the pendant group is a peptide.
 7. The composition of claim 6,wherein the bioactive agent is a cyclic Arg-Gly-Asp (cRGD) peptide. 8.The composition of claim 1, wherein the bioactive agent is an ioniccrosslinking agent.
 9. The composition of claim 8, wherein the bioactiveagent is Ca²⁺, Ba²⁺, or Sr²⁺. 10-13. (canceled)
 14. A compositioncomprising a PPCN-based peptide-displaying hydrogel comprising citricacid, poly(ethylene glycol), glycerol 1,3-diglycerolate diacrylate, andpoly-(N-isopropylacrylamide) monomers, and a peptide covalentlyconjugated to carboxy groups of the citric acid monomers.
 15. Thecomposition of claim 14, wherein the peptide is covalently conjugatedvia carbodiimide chemistry to carboxy groups of the citric acidmonomers.
 16. The composition of claim 14, wherein the peptide is cyclicArg-Gly-Asp (cRGD).
 17. The composition of claim 16, wherein the cRGD iscovalently conjugated to carboxy groups of PPCN.
 18. A compositioncomprising a PPCN-based hydrogel comprising citric acid, poly(ethyleneglycol), glycerol 1,3-diglycerolate diacrylate, andpoly-(N-isopropylacrylamide) monomers, and metal ion crosslinks.
 19. Thecomposition of claim 18, comprising metal-ion-crosslinked PPCN.
 20. Thecomposition of claim 19, prepared by incubating the PPCN in the presenceof a salt of the metal ion.
 21. The composition of claim 19, wherein themetal ion is Ca²⁺, Ba²⁺, or Sr²⁺.
 22. A method of facilitating bonerepair comprising administering a composition of claim 1 to fractured ordiseased bone site, and allowing the composition to gel.
 23. (canceled)